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The VRC01 Antibody Mediated Prevention (AMP) efficacy trials conducted between 2016 and 2020 showed for the first time that passively administered broadly neutralizing antibodies (bnAbs) could prevent HIV-1 acquisition against bnAb-sensitive viruses. HIV-1 viruses isolated from AMP participants who acquired infection during the study in the sub-Saharan African (HVTN 703/HPTN 081) and the Americas/European (HVTN 704/HPTN 085) trials represent a panel of currently circulating strains of HIV-1 and offer a unique opportunity to investigate the sensitivity of the virus to broadly neutralizing antibodies (bnAbs) being considered for clinical development. Pseudoviruses were constructed using envelope sequences from 218 individuals. The majority of viruses identified were clade B and C; with clades A, D, F and G and recombinants AC and BF detected at lower frequencies. We tested eight bnAbs in clinical development (VRC01, VRC07-523LS, 3BNC117, CAP256.25, PGDM1400, PGT121, 10–1074 and 10E8v4) for neutralization against all AMP placebo viruses (n = 76). Compared to older clade C viruses (1998–2010), the HVTN703/HPTN081 clade C viruses showed increased resistance to VRC07-523LS and CAP256.25. At a concentration of 1μg/ml (IC80), predictive modeling identified the triple combination of V3/V2-glycan/CD4bs-targeting bnAbs (10-1074/PGDM1400/VRC07-523LS) as the best against clade C viruses and a combination of MPER/V3/CD4bs-targeting bnAbs (10E8v4/10-1074/VRC07-523LS) as the best against clade B viruses, due to low coverage of V2-glycan directed bnAbs against clade B viruses. Overall, the AMP placebo viruses represent a valuable resource for defining the sensitivity of contemporaneous circulating viral strains to bnAbs and highlight the need to update reference panels regularly. Our data also suggests that combining bnAbs in passive immunization trials would improve coverage of global viruses.

Biological sex differences affect the course of HIV infection, with untreated women having lower viral loads compared to their male counterparts but, for a given viral load, women have a higher rate of progression to AIDS. However, the vast majority of data on viral evolution, a process that is clearly impacted by host immunity and could be impacted by sex differences, has been derived from men. We conducted an intensive analysis of HIV-1 gag and env-gp120 evolution taken over the first 6-11 years of infection from 8 Women's Interagency HIV Study (WIHS) participants who had not received combination antiretroviral therapy (ART). This was compared to similar data previously collected from men, with both groups infected with HIV-1 subtype B. Early virus populations in men and women were generally homogenous with no differences in diversity between sexes. No differences in ensuing nucleotide substitution rates were found between the female and male cohorts studied herein. As previously reported for men, time to peak diversity in env-gp120 in women was positively associated with time to CD4+ cell count below 200 (P = 0.017), and the number of predicted N-linked glycosylation sites generally increased over time, followed by a plateau or decline, with the majority of changes localized to the V1-V2 region. These findings strongly suggest that the sex differences in HIV-1 disease progression attributed to immune system composition and sensitivities are not revealed by, nor do they impact, global patterns of viral evolution, the latter of which proceeds similarly in women and men.

In HIV prevention trials, accurate timing estimates of when individual participants acquire HIV can be used to estimate antibody levels at the time of acquisition, which is useful for projecting antibody levels needed for prevention. The results we report here suggest that if sequence-based estimation of acquisition timing is used in future clinical trials of combination broadly neutralizing antibody (bnAb) regimens or multispecific bnAbs for HIV prevention, a sampling frequency of at least monthly is needed. Moreover, in the samples analyzed here, we observed less bias in sequence-based timing estimation for samples taken <5 weeks post-DDA. This observation is consistent with the timing of immune-driven selective pressures that may negatively impact the power to detect acquisition sieve effects.

In the antibody mediated prevention (AMP) trials, the broadly neutralizing antibody (bNAb) VRC01 demonstrated protective efficacy against susceptible HIV strains. To understand how VRC01 shaped breakthrough infections, deep sequencing was performed on 172 participants (>100,000 gag-Δpol and rev-env-Δnef sequences), at diagnosis and over time, in the placebo and treatment arms of the African (HVTN703/HPTN081; NCT02568215) and Americas/Europe (HVTN704/HPTN085; NCT02716675) cohorts. A high frequency of multilineage infections was detected (38%), including co-infection with both VRC01 sensitive and resistant viruses. This high frequency is largely accounted for by low-abundance lineages. Although VRC01 does not significantly affect the genetic transmission bottleneck compared to placebo, higher VRC01 doses trend towards greater VRC01 neutralization differences among co-infecting lineages. Two-thirds of multilineage infections showed evidence of recombination at the diagnostic timepoint. In the treatment group there is evidence of recombinant viruses preferentially inheriting resistance-associated mutations. This study provides critical insights into viral genetic and antigenic diversity that needs to be targeted to achieve protection, and highlights the role of recombination in facilitating escape. Antibody mediated prevention (AMP) trials with the broadly neutralizing antibody VRC01 showed protection against VRC01-sensitive viruses. Here, by deep sequencing plasma samples from 172 participants of the AMP trials, the authors show a high frequency of multilineage HIV infections (38%), including coinfection with both sensitive and resistant viruses, and demonstrate that VRC01 doesn’t alter the transmission bottleneck.

Understanding the selective forces acting upon HIV early in infection is crucial to design prevention strategies. By leveraging deep sequencing and the short diagnostic intervals of the FRESH and RV217 cohorts between the last-negative and first-positive RNA tests (median 4 days), we captured a precise and early snapshot of acute HIV infection. The frequency of multiple transmitted viruses of 37% in these as well as placebo recipients from the AMP trials (NCT02716675 and NCT02568215) was higher than previously published, with the true frequency likely to be higher. The relative abundance of lineages fluctuated substantially over time in two-thirds of the multilineage infections, generating uncertainty in identifying the specific viruses that were transmitted and founding the infection. At the population level, viral populations exhibited limited diversity and selection on the Gag and Env proteins at the earliest times examined, with sites inferred to be undergoing negative selection most evident. These data may help explain vaccination failures and provide new targets for prevention.

Abstract Pathogen diversity resulting in quasispecies can enable persistence and adaptation to host defenses and therapies. However, accurate quasispecies characterization can be impeded by errors introduced during sample handling and sequencing, which can require extensive optimizations to overcome. We present complete laboratory and bioinformatics workflows to overcome many of these hurdles. The Pacific Biosciences single molecule real-time platform was used to sequence polymerase-chain reaction (PCR) amplicons derived from cDNA templates tagged with unique molecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR. The use of UMI allowed accurate template quantitation as well as removal of point mutations introduced during PCR and sequencing to produce a highly accurate consensus sequence from each template. Production of highly accurate sequences from the large datasets produced from SMRT-UMI sequencing is facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline). PORPIDpipeline automatically filters and parses circular consensus reads by sample, identifies and discards reads with UMIs likely created from PCR and sequencing errors, generates consensus sequences, checks for contamination within the dataset, and removes any sequence with evidence of PCR recombination, heteroduplex formation, or early cycle PCR errors. The optimized SMRT-UMI sequencing and PORPIDpipeline methods presented here represent a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus quasispecies in a virus transmitter-recipient pair of individuals.

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma (KS), yet the viral genetic factors that lead to the development of KS in KSHV-infected individuals have not been fully elucidated. Nearly, all previous analyses of KSHV genomic evolution and diversity have excluded the three major internal repeat regions: the two origins of lytic replication, internal repeats 1 and 2 (IR1 and IR2), and the latency-associated nuclear antigen (LANA) repeat domain (LANAr). These regions encode protein domains that are essential to the KSHV infection cycle but have been rarely sequenced due to their extended repetitive nature and high guanine and cytosine (GC) content. The limited data available suggest that their sequences and repeat lengths are more heterogeneous across individuals than in the remainder of the KSHV genome. To assess their diversity, the full-length IR1, IR2, and LANAr sequences, tagged with unique molecular identifiers (UMIs), were obtained by Pacific Biosciences’ single-molecule real-time sequencing (SMRT-UMI) from twenty-four tumors and six matching oral swabs from sixteen adults in Uganda with advanced KS. Intra-host single-nucleotide variation involved an average of 0.16 per cent of base positions in the repeat regions compared to a nearly identical average of 0.17 per cent of base positions in the remainder of the genome. Tandem repeat unit (TRU) counts varied by only one from the intra-host consensus in a majority of individuals. Including the TRU indels, the average intra-host pairwise identity was 98.3 per cent for IR1, 99.6 per cent for IR2 and 98.9 per cent for LANAr. More individuals had mismatches and variable TRU counts in IR1 (twelve/sixteen) than in IR2 (two/sixteen). There were no open reading frames in the Kaposin coding sequence inside IR2 in at least fifty-five of ninety-six sequences. In summary, the KSHV major internal repeats, like the rest of the genome in individuals with KS, have low diversity. IR1 was the most variable among the repeats, and no intact Kaposin reading frames were present in IR2 of the majority of genomes sampled.

Broadly neutralizing antibodies (bNAbs) show promise for HIV treatment and prevention, but are vulnerable to resistance evolution. Comprehensively understanding viral escape from individual bNAbs is necessary to design bNAb combinations that will provide durable responses. We characterize viral escape from two such bNAbs, 10-1074 and 3BNC117, using deep, longitudinal sequencing of full length HIV envelope ( ) genes from study participants treated with bNAb monotherapy. Improved sequencing depth and computational evolutionary analyses permit us to identify routes and parallelism underlying HIV escape from each bNAb, providing new insights into this evolutionary process: 10-1074 escape is restricted to a small number of previously documented pathways, but these escape mutations 1) pre-exist in intra-host viral populations before therapy, 2) are not all equally preferred, and 3) emerge with a high degree of genetic parallelism within and across viral populations. In contrast, 3BNC117 escape follows background-specific patterns in which specific escape mutations present in one population rarely emerge or spread in other populations, but often still exhibit parallel evolutionary responses within their host. That bNAbs elicit starkly different escape profiles depending on their Env target exposes the limitations of generalizing escape patterns across therapies and highlights the substantial challenges in predicting a viral population's bNAb susceptibility from genetic diversity alone.

HIV-1 cure requires preventing viral rebound after treatment interruption, but quantitative criteria defining the rebound-competent reservoir are lacking. We studied individuals undergoing observational treatment interruption to identify virologic and immunologic determinants of rebound. In 9 of 13 participants, rebound viruses were genetically identical or similar to proviruses in circulating resting CD4⁺ T-cells. We found no evidence of recombination among rebound sequences, rather resistance to autologous neutralizing antibodies was a critical determinant of viral rebound. Using inhibitory potential ( ), the log reduction in single-round infection at physiologic IgG concentrations, we defined quantitative limits governing rebound-competency. Reservoir variants exhibited a wide range of values (0.4-8.2 logs), whereas rebound viruses were minimally inhibited (0.5-2.8 logs), indicating that inhibition by even up to 2.8 logs (631-fold) cannot prevent rebound. Longitudinal analyses revealed that waning aNAb potency allows previously neutralized variants to gain rebound potential. Thus, rebound competency is a dynamic, immune-governed property defined by quantitative immunologic constraints.